Molecular Biological and Immunohistochemical Study of Differentiation of the Osteoblast.

在这笔赠款支持的研究中，明确了以下结果1。用Northern杂交方法研究了BGP和MGP基因在大鼠成骨细胞系C11、C20、C23和C26中的表达。所有细胞株均表达MGP基因，但不表达BGP基因。结论：1.加入L、25-二羟基维生素D_3后，除C26细胞处于成骨分化的未成熟阶段外，其余细胞的Mgp基因表达水平均显著升高。在与Genetics Institute Inc.的合作研究中，我们发现重组人骨形态发生蛋白-2(BMP-2)可以刺激C26细胞的成骨成熟。分别从小鼠成骨细胞系MC3T3-EL和细胞系C20构建的cDNA文库中克隆了小鼠和大鼠MGP的cDNAs。核苷酸测序结果表明，克隆的小鼠MGP cDNAs中有五个谷氨酸残基中的一个可能修饰为GLA不保守。为了研究成骨细胞在骨组织中的分化，我们建立了不脱钙骨组织的原位杂交新方法。利用该方法，我们研究了BGP和MGP基因在成年大鼠股骨和腰部的表达。BGP基因在矿化骨槽中的立方成骨细胞中表达较强。从另一方面来说。MGP基因在肥大的软骨细胞中呈强阳性表达。根据这些结果，BGP和MGP基因在矿化前在未成熟骨组织中的表达被认为与在成熟骨组织中的表达不同。

In the studies supported by this grant, the following results were cleared.1. The expression of BGP and MGP genes in the rat osteoblastic cell lines, C11, C20, C23 and C26 was studied by the method of Northern blot hybridization. All the cell lines expressed MGP gene, but did not express BGP gene. After administration of l,25-dihydroxyvitamin D_3, the levels of MGP gene expression was greatly increased except for C26 cells, which are in more immature stage of osteoblastic differentiation than the other cell lines.2. We found that recombinant human bone morphogenetic protein-2 (BMP-2) stimulates osteoblastic maturation of C26 cells in the collaboration study with Genetics Institute Inc.3. The mouse and rat MGP cDNAs were cloned from the cDNA libraries constructed from mouse osteoblastic cell line MC3T3-El, and the cell line C20, respectively. As a result of the nucleotide sequencing, the cloned mouse MGP cDNAs were found that one of the five glutamic acid residues potentially modified to Gla not conserved.4. To study differentiation of osteoblast in the bone tissue, wo developed the new method for in situ hybridization of non-decalcified bone tissue. Using this method, we studied the expression of BGP and MGP genes in the femur and lumbar of adukt rats. BGP gene was strongly expressed in cuboidal osteoblasts along the mineralized bone traveculars. On the other hand. MGP gene was expressed strongly in hypertrophic chondrocytes. From these results, the expression of BGP and MGP genes in immature bone tissue before mineralization was thought to be different from the expression in mature bone tissue.